In fluorescence microscopy, fluorescent dye particles in a sample are excited to fluoresce. The dye particles in the sample are bound to molecules of the sample, so that detection of the fluorescent light allows conclusions to be drawn about structures and processes in the sample. The fluorescent dye particles are also referred to as marker substances or markers. The fluorescent dye particles are either naturally present in the sample, or artificially incorporated into the sample and coupled to the molecules of the sample.
Some fluorescence microscopes are capable of imaging structures in a sample which are smaller than the diffraction resolution limit of conventional light microscopes. Furthermore, these fluorescence microscopes are able to image processes taking place in an area smaller than the diffraction resolution limit of conventional light microscopes. These fluorescence microscopes are based on sequential, stochastic localization of dye particles. The dye particles have two distinguishable states. In a first active state, the dye particles can be excited to fluoresce, while in a second inactive state, the dye particles cannot be excited to fluoresce. Moreover, the dye particles can be transferred from the active to the inactive state, or from the inactive to the active state.
In order to overcome the resolution limit imposed by diffraction, a large portion of the dye particles are transferred to the inactive state, or only a small fraction are transferred to the active state, so that, as a result, only a relatively small fraction of the dye particles are in the active state. Switching from the active state to the inactive state, or from the inactive state to the active state, can be accomplished in different ways.
International Publication WO 2006/12769 A2 describes a switching process from an active state to an inactive state, and then from an inactive state to an active state. In particular, dye particles are used which can be transferred from the inactive state to the active state by irradiation with light of a defined activation wavelength. A portion of the dye particles in the active state can be returned to the inactive state by bleaching, which further reduces the subset of active dye particles. Subsequently, the remaining active dye particles of the subset are excited to fluoresce by the excitation light.
In the publication Appl. Phys. A, 88, 223-226, 2007, a method is described which uses dye particles capable of being reversibly transferred from the inactive state to the active state by irradiation with light of a defined activation wavelength, and of being reversibly returned from the active state to the inactive state by irradiation with light of a defined deactivation wavelength. The active dye particles are excited to fluoresce by the excitation light.
German Publication DE 10 2008 024 568 A1 describes the use of dye particles which have transient dark states, such as triplet states. A large portion of these dye particles are transferred to the dark state, and automatically return to the active state with a defined probability after a residence time which is dependent on the type of molecule.
The methods described in the above-mentioned documents are known under the names of PALM, FPALM, (F)STORM, PALMIRA, dSTORM and GSDIM. All these methods have in common that only a subset of dye particles is transferred to the active state and excited to fluoresce while in the active state. The subset of active dye particles must be so small that the average distance between neighboring dye particles in the active state is greater than the conventional resolution limit of the imaging optical system. The fluorescent light from the subset of active dye particles is imaged onto a spatially resolving photodetector, such as a CCD camera, in particular an EM-CCD camera. The use of a spatially resolving photodetector makes it possible to then display a graphical representation of the fluorescent light distribution which is representative of a distribution of the fluorescent dye particles in the sample. In particular, the graphical representation of the light distribution exhibits light spots whose size is determined by the unsharpness of the imaging optical system and which are representative of the dye particles in the sample. For each of the light spots, a comparison value representative of a light quantity causing the light sport is determined using known algorithms. If the comparison value is greater than a predefined threshold value, then the light spot is classified as an event. Subsequently, additional images are captured, which show further light spots, at least a portion of which are then classified as events. Then, an image of the searched structures or processes in the sample is generated based on all events. The threshold value may also be referred to as evaluation parameter. The comparison value may be, for example, a light quantity, a light intensity, a light energy, or a luminance within the subregion of the graphical representation of the light distribution that causes the light spot. The selection of a suitable evaluation parameter is decisive for the quality of the final image.